Detection of ER stress in vivo by Raman spectroscopy

The endoplasmic reticulum (ER) is an organelle in which most membrane and secretory proteins are synthesized. If these proteins are not folded correctly, unfolded proteins accumulate in the ER lumen, causing a cellular situation known as ER stress. Recently, many studies on the relationship between ER stress and diseases have been reported. Thus, studies of ER stress in vivo should yield information that is useful in pathology. Model mice have been developed as a powerful tool to visualize ER stress in vivo, but this approach depends on transgenic technology. Here, we report on a method of detecting ER stress in vivo by Raman spectroscopy. Our experiments revealed that two specific Raman bands were reduced in both cultured cells and animal tissues in an ER stress dependent manner. This suggests that Raman spectroscopy could be a useful tool to detect ER stress in vivo without transgenic technology.     

Cloning and characterization of VIGG,a novel virus-induced grapevine protein,correlated with fruit quality

We report here the identification and characterization of VIGG, a novel virus-induced grapevine protein. Analysis of VIGG expression in grapevine demonstrated that VIGG was constitutively expressed in leaves and stems in virus-infected grapevine, and that VIGG expression was induced by grapevine virus A (GVA) infection, but not by infection with other viruses. The virus-induced expression profile of VIGG was supported by the finding that virus-free meristem cultures prepared from virus-infected grapevines did not express VIGG. An experiment using GFP–VIGG fusion protein demonstrated that VIGG might be localized in or around the endoplasmic reticulum (ER). Treatment of grapevine cells with ER stress inducers resulted in the induction of VIGG expression. Berries from VIGG-expressing grapevines had higher organic acid and phenolic contents than those from control grapevines that did not express VIGG. Interestingly, fruit composition of a grapevine that was simultaneously infected by GVA and grapevine virus B (GVB), which did not express VIGG, was significantly different from that of GVA-infected grapevines expressing VIGG, suggesting that the effector of fruit composition alteration might be VIGG expression, but not GVA infection. Taken together, VIGG expression might suppress the decrease in organic acid content and increase phenol content in berries. Further investigation of the biological function of VIGG is expected to provide new information on the fruit quality of grapevines.     

Bcl-2 proteins regulate ER membrane permeability to luminal proteins during ER stress-induced apoptosis

Endoplasmic reticulum (ER) stress-induced apoptosis may arise from multiple environmental and pharmacological causes, but the precise mechanism(s) involved are not completely known. Members of Bcl-2 protein family are important regulators of apoptosis. In this study, we report that in a process dependent on the proapoptotic Bcl-2 members Bax and Bak, exogenously expressed fluorescent protein localized to the ER lumen is released into the cytosol in cells undergoing ER stress. Upon ER stress induction, endogenous ER luminal proteins are also released into the cytosol in a similar manner accompanied by translocation and anchorage of Bax to the ER membrane. In addition, Bax and truncated-Bid (tBid) mediate a global increase in ER membrane permeability to ER luminal proteins in vitro. Importantly, antiapoptotic Bcl-X_L antagonizes the effects of proapoptotic Bcl-2 proteins on ER membrane permeability. Consistent with Bax translocation to the ER membrane in whole apoptotic cells, there is also increased tight association of Bax with the ER membrane correlated with the increase in ER membrane permeability in vitro. Overall, these data suggest that the regulation of ER membrane permeability by Bcl-2 proteins could be an important molecular mechanism of ER stress-induced apoptosis.     

An acyl-CoA-binding protein from grape that is induced through ER stress confers morphological changes and disease resistance in Arabidopsis

We here report characterization of a grape (Vitis vinifera) acyl-CoA-binding protein (VvACBP). Expression of VvACBP was detected in grape leaves exposed to tunicamycin-induced endoplasmic reticulum (ER) stress as well as cold and heat shock treatments. In tendrils and peduncles, however, high-temperature treatment induced BiP (luminal binding protein) expression, a marker of ER stress in berry skin, but not VvACBP expression. We hypothesize that VvACBP may be sorted to the periphery of plant cells. Transgenic Arabidopsis plants, expressing VvACBP, exhibited slowed-down floral transition. The gene expression of proteins related to the photoperiodic pathway, CONSTANS, FLOWERING LOCUS T (FT), and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), was down-regulated in transgenic seedlings. These results underscore the possibility that VvACBP may affect the regulation of floral transition in Arabidopsis by suppressing the photoperiodic pathway. The transgenic Arabidopsis plants also exhibited morphological changes such as thicker inflorescences and rosette leaves. In addition, the rosette leaves of the transgenic plants had higher anthocyanin, total phenol, and chlorophyll contents than those of the control plants. Finally, the transgenic plants showed disease resistance to Pseudomonas syringae and Colletotrichum higginsianum, suggesting that VvACBP may also enhance disease resistance in grapevine.     

Jasmonic acid‐inducible TSA1 facilitates ER body formation

Members of the Brassicales contain an organelle, the endoplasmic reticulum (ER) body, which is derived from the ER. Recent studies have shed light on the biogenesis of the ER body and its physiological role in plants. However, formation of the ER body and its physiological role are not fully understood. Here, we investigated the physiological role of TSK‐associating protein 1 (TSA1), a close homolog of NAI2 that is involved in ER body formation, and provide evidence that it is involved in ER body biogenesis under wound‐related stress conditions. TSA1 is N‐glycosylated and localizes to the ER body as a luminal protein. TSA1 was highly induced by the plant hormone, methyl jasmonate (MeJA). Ectopic expression of TSA1:GFP induced ER body formation in root tissues of transgenic Arabidopsis thaliana and in leaf tissues of Nicotiana benthamiana. TSA1 and NAI2 formed a heterocomplex and showed an additive effect on ER body formation in N. benthamiana. MeJA treatment induced ER body formation in leaf tissues of nai2 and tsa1 plants, but not nai2/tsa1 double‐mutant plants. However, constitutive ER body formation was altered in young seedlings of nai2 plants but not tsa1 plants. Based on these results, we propose that TSA1 plays a critical role in MeJA‐induced ER body formation in plants.     

ER chaperones in mammalian development and human diseases

The field of endoplasmic reticulum (ER) stress in mammalian cells has expanded rapidly during the past decade, contributing to understanding of the molecular pathways that allow cells to adapt to perturbations in ER homeostasis. One major mechanism is mediated by molecular ER chaperones which are critical not only for quality control of proteins processed in the ER, but also for regulation of ER signaling in response to ER stress. Here, we summarized the properties and functions of GRP78/BiP, GRP94/gp96, GRP170/ORP150, GRP58/ERp57, PDI, ERp72, calnexin, calreticulin, EDEM, Herp and co-chaperones SIL1 and P58(IPK) and their role in development and diseases. Many of the new insights are derived from recently constructed mouse models where the genes encoding the chaperones are genetically altered, providing invaluable tools for examining the physiological involvement of the ER chaperones in vivo.     

Isolation of proteins that speifically interact with the ATPase domain of mammalian ER chaperone,BiP

BiP, immunoglobulin binding protein, is an ER homologue of Hsp 70. However, unlike other Hsp70 proteins, regulatory protein(s) for BiP has not been identified. Here, we demonstrated the presence of potential regulatory proteins for BiP using a pull-down assay. Since BiP can bind any unfolded protein, only the ATPase domain of BiP was used for the pull-down assay in order to minimize nonspecific binding. The ATPase domain was cloned to produce recombinant protein, which was then conjugated to CNBr-activated agarose. The structural conformation and ATP hydrolysis activity of the recombinant ATPase domain were similar to those of the native protein. Eight proteins from metabolically labeled mouse plasmacytoma cells specifically bound to the recombinant ATPase protein. The binding of these proteins was inhibited by excess amounts of free ATPase protein, and was dependent on the presence of ATP. These proteins were eluted by ADP. Of these proteins, Grp 170 and BiP where identified, while the others were not identified as known ER proteins, from Western blot analyses. The presence of the ATPase-binding proteins for Bip was first demonstrated in this study, and our data suggest similar regulatory machinery for BiP may exist in the ER, as found in prokaryotes and other cellular compartments.     

The endoplasmic reticulum-associated degradation is necessary for plant salt tolerance

Eukaryotic organisms have quality-control mechanisms that allow misfolded or unassembled proteins to be retained in the endoplasmic reticulum (ER) and subsequently degraded by ER-associated degradation (ERAD). The ERAD pathway is well studied in yeast and mammals; however, the biological functions of plant ERAD have not been reported. Through molecular and cellular biological approaches, we found that ERAD is necessary for plants to overcome salt stress. Upon salt treatment ubiquitinated proteins increased in plant cells, especially unfolded proteins that quickly accumulated in the ER and subsequently induced ER stress responses. Defect in HRD3A of the HRD1/HRD3 complex of the ERAD pathway resulted in alteration of the unfolded protein response (UPR), increased plant sensitivity to salt, and retention of ERAD substrates in plant cells. Furthermore, we demonstrated that Ca(2+) release from the ER is involved in the elevation of UPR and reactive oxygen species (ROS) participates the ERAD-related plant salt response pathway.     

疱疹病毒与内质网应激

内质网是分泌型蛋白和膜蛋白折叠及翻译后修饰的主要场所.病毒感染所引起的宿主细胞内环境的改变可使细胞或病毒的未折叠和/或错误折叠蛋白在内质网中大量聚集,使内质网处于生理功能紊乱的应激状态.为了缓解这种应激压力,细胞会启动未折叠蛋白反应(UPR),并通过一系列分子的信号转导维持内质网稳态;同时病毒也会通过对UPR的精密调控...     

Degradation of the endoplasmic reticulum by autophagy in plants

Eukaryotic cells have developed sophisticated strategies to contend with environmental stresses faced in their lifetime. Endoplasmic reticulum (ER) stress occurs when the accumulation of unfolded proteins within the ER exceeds the folding capacity of ER chaperones. ER stress responses have been well characterized in animals and yeast, and autophagy has been suggested to play an important role in recovery from ER stress. In plants, the unfolded protein response signaling pathways have been studied, but changes in ER morphology and ER homeostasis during ER stress have not been analyzed previously. Autophagy has been reported to function in tolerance of several stress conditions in plants, including nutrient deprivation, salt and drought stresses, oxidative stress, and pathogen infection. However, whether autophagy also functions during ER stress has not been investigated. The goal of our study was to elucidate the role and regulation of autophagy during ER stress in Arabidopsis thaliana.     

Endoplasmic reticulum proteostasis impairment in aging

Perturbed neuronal proteostasis is a salient feature shared by both aging and protein misfolding disorders. The proteostasis network controls the health of the proteome by integrating pathways involved in protein synthesis, folding, trafficking, secretion, and their degradation. A reduction in the buffering capacity of the proteostasis network during aging may increase the risk to undergo neurodegeneration by enhancing the accumulation of misfolded proteins. As almost one‐third of the proteome is synthetized at the endoplasmic reticulum (ER), maintenance of its proper function is fundamental to sustain neuronal function. In fact, ER stress is a common feature of most neurodegenerative diseases. The unfolded protein response (UPR) operates as central player to maintain ER homeostasis or the induction of cell death of chronically damaged cells. Here, we discuss recent evidence placing ER stress as a driver of brain aging, and the emerging impact of neuronal UPR in controlling global proteostasis at the whole organismal level. Finally, we discuss possible therapeutic interventions to improve proteostasis and prevent pathological brain aging.     

Rab7a modulates ER stress and ER morphology

The Endoplasmic Reticulum (ER) is a membranous organelle with diverse structural and functional domains. Peripheral ER includes interconnected tubules, and dense tubular arrays called “ER matrices” together with bona fide flat cisternae. Transitions between these states are regulated by membrane-associated proteins and cytosolic factors. Recently, the small GTPases Rab10 and Rab18 were reported to control ER shape by regulating ER dynamics and fusion. Here, we present evidence that another Rab protein, Rab7a, modulates the ER morphology by controlling the ER homeostasis and ER stress. Indeed, inhibition of Rab7a expression by siRNA or expression of the dominant negative mutant Rab7aT22?N, leads to enlargement of sheet-like ER structures and spreading towards the cell periphery. Notably, such alterations are ascribable neither to a direct modulation of the ER shaping proteins Reticulon-4b and CLIMP63, nor to interactions with Protrudin, a Rab7a-binding protein known to affect the ER organization. Conversely, depletion of Rab7a leads to basal ER stress, in turn causing ER membrane expansion. Both ER enlargement and basal ER stress are reverted in rescue experiments by Rab7a re-expression, as well as by the ER chemical chaperone tauroursodeoxycholic acid (TUDCA). Collectively, these findings reveal a new role of Rab7a in ER homeostasis, and indicate that genetic and pharmacological ER stress manipulation may restore ER morphology in Rab7a silenced cells.     

Zinc and the Msc2 zinc transporter protein are required for endoplasmic reticulum function

In this report, we show that zinc is required for endoplasmic reticulum function in Saccharomyces cerevisiae. Zinc deficiency in this yeast induces the unfolded protein response (UPR), a system normally activated by unfolded ER proteins. Msc2, a member of the cation diffusion facilitator (CDF) family of metal ion transporters, was previously implicated in zinc homeostasis. Our results indicate that Msc2 is one route of zinc entry into the ER. Msc2 localizes to the ER when expressed at normal levels. UPR induction in low zinc is exacerbated in an msc2 mutant. Genetic and biochemical evidence indicates that this UPR induction is due to genuine ER dysfunction. Notably, we found that ER-associated protein degradation is defective in zinc-limited msc2 mutants. We also show that the vacuolar CDF proteins Zrc1 and Cot1 are other pathways of ER zinc acquisition. Finally, zinc deficiency up-regulates the mammalian ER stress response indicating a conserved requirement for zinc in ER function among eukaryotes.     

The plant-unique cis-element that mediates signaling from multiple endoplasmic reticulum stress sensors

The accumulation of unfolded proteins in the ER lumen induces intracellular signaling mediated by the ER stress sensor protein IRE1. Our recent study identified a new common cis-element of ER stress-responsive genes (such as rice BiP paralogs and WRKY45) that were regulated via an IRE1-dependent pathway. ER stress-responsive cis-elements had been expected to be conserved between plants and mammals. However, contrary to expectations, sequences of the plant cis-element, pUPRE-II, were not identical to those of its mammalian counterpart. Additionally, pUPRE-II also interacted with another ER stress sensor protein and mediated multiple signaling pathways. Here, we provide a summary of the results that suggest the complicated mechanism underlying the regulation of ER stress-responsive gene expression in plants.     

Endoplasmic reticulum: ER stress regulates mitochondrial bioenergetics

Endoplasmic reticulum (ER) stress activates an adaptive unfolded protein response (UPR) that facilitates cellular repair, however, under prolonged ER stress, the UPR can ultimately trigger apoptosis thereby terminating damaged cells. The molecular mechanisms responsible for execution of the cell death program are relatively well characterized, but the metabolic events taking place during the adaptive phase of ER stress remain largely undefined. Here we discuss emerging evidence regarding the metabolic changes that occur during the onset of ER stress and how ER influences mitochondrial function through mechanisms involving calcium transfer, thereby facilitating cellular adaptation. Finally, we highlight how dysregulation of ER-mitochondrial calcium homeostasis during prolonged ER stress is emerging as a novel mechanism implicated in the onset of metabolic disorders.     

未折叠蛋白响应的激活机制

未折叠蛋白在内质网（endoplasmic reticulum,ER）腔中累积造成ER应激，此时细胞启动未折叠蛋白响应（unfolded protein response,UPR）以恢复蛋白质稳态。目前已知有三种UPR感受器，即IRE1、PERK和ATF6，它们均为ER跨膜蛋白，在ER应激时被激活并启动下游UPR信号通路。虽然UPR感受器最早是在研究细胞如何应对ER应激时发现的，但它们如何感知ER应激至今未得到完满的回答。随着研究的深入，人们发现UPR的功能不仅限于维持蛋白质稳态，而UPR感受器也不是只对未折叠蛋白累积作出响应。本文对UPR的发现及其经典通路作一介绍，着重阐述目前已知的UPR感受器的激活机制，并就UPR和ER应激关系以及该领域存在的问题进行讨论。     

In tobacco leaf epidermal cells, the integrity of protein export from the endoplasmic reticulum and of ER export sites depends on active COPI machinery

Trafficking of secretory proteins between the endoplasmic reticulum (ER) and the Golgi apparatus depends on coat protein complexes I (COPI) and II (COPII) machineries. To date, full characterization of the distribution and dynamics of these machineries in plant cells remains elusive. Furthermore, except for a presumed linkage between COPI and COPII for the maintenance of ER protein export, the mechanisms by which COPI influences COPII-mediated protein transport from the ER in plant cells are largely uncharacterized. Here we dissect the dynamics of COPI in intact cells using live-cell imaging and fluorescence recovery after photobleaching analyses to provide insights into the distribution of COPI and COPII machineries and the mechanisms by which COPI influences COPII-mediated protein export from the ER. We found that Arf1 and coatomer are dynamically associated with the Golgi apparatus and that the COPII coat proteins Sec24 and Sec23 localize at ER export sites that track with the Golgi apparatus in tobacco leaf epidermal cells. Arf1 is also localized at additional structures that originate from the Golgi apparatus but that lack coatomer, supporting the model that Arf1 also has a coatomer-independent role for post-Golgi protein transport in plants. When ER to Golgi protein transport is inhibited by mutations that hamper Arf1-GTPase activity without directly disrupting the COPII machinery for ER protein export, Golgi markers are localized in the ER and the punctate distribution of Sec24 and Sec23 at the ER export sites is lost. These findings suggest that Golgi membrane protein distribution is maintained by the balanced action of COPI and COPII systems, and that Arf1-coatomer is most likely indirectly required for forward trafficking out of the ER due to its role in recycling components that are essential for differentiation of the ER export domains formed by the Sar1-COPII system.     

Inhibition of PKR protects against tunicamycin-induced apoptosis in neuroblastoma cells

Endoplasmic reticulum (ER) dysfunction is thought to play a significant role in several neurological disorders, including Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, amyotrophic lateral sclerosis, cerebral ischemia, and the prion diseases. ER dysfunction can be mimicked by cellular stress signals such as disruption of calcium homeostasis, inhibition of protein glycosylation, and reduction of disulfide bonds, which results in accumulation of misfolded proteins in the ER and leads to cell death by apoptosis. Tunicamycin, which is an inhibitor of protein glycosylation, induces ER stress and apoptosis. In this study, we examined the involvement of double stranded (ds) RNA-activated protein kinase PKR in tunicamycin-induced apoptosis. We used overexpression of the trans-dominant negative, catalytically inactive mutant K296R to inhibit PKR activity in neuroblastoma cells. We demonstrate that inhibition of PKR activation in response to tunicamycin protects neuronal cells from undergoing apoptosis. Furthermore, K296R overexpressing cells show defective PKR activation, delayed eIF2α phosphorylation, dramatically delayed ATF4 expression. In addition, both caspase-3 activation and C/EBP homologous protein (CHOP, also known as GADD153) induction, which are markers of apoptotic cells, are absent from K296R overexpression cells in response to tunicamycin. These results establish that PKR activation plays a major regulatory role in induction of apoptosis in response to ER stress and indicates the potential of PKR as possible target for neuroprotective therapeutics.